Fig. 3: TKS4/5 and MT1-MMP are internalized into intraluminal vesicles of late endosomes and co-secreted in extracellular vesicles.
From: Intercellular transfer of cancer cell invasiveness via endosome-mediated protease shedding
a MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells were grown on cover slips, fixed and immunostained with antibodies against TKS5, mCh and LAMP1. Samples were analysed by superresolution microscopy (Airyscan). Maximum intensity projection of a z-stack and Imaris surface 3D renderings are shown, visualizing that TKS5-GFP and mCh-MT1-MMP appear to localise inside endosomes. Representative of 10 images of 19 cells. b Immunogold labelling with silver enhancement of MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells with antibodies against MT1-MMP or GFP. Electron microscopy images show labelling of mCh-MT1-MMP (left column) and TKS5-GFP (right column) inside the lumen of MVEs. The arrows point to immunogold-labelling-positive ILVs. Representative of 21 (anti-MT1-MMP) and 28 (anti-GFP) MVEs. c MDA-MB-231 parental or mCh-MT1-MMP stably over-expressing cells were used to isolate extracellular vesicles (EVs) using ultracentrifugation (100 000xg) as described in the methods section. Western blots (WB) and graphs show the presence of the indicated proteins in whole cell lysate (WCL) normalized to vinculin or GAPDH, and EV fraction normalized to TSG101 (TKS4) and to CD9 (TKS5). * MT1-MMP variants likely comprising processed forms of MT1-MMP and mCh-MT1-MMP (immature, posttranslationally modified or cleaved). Error bars denote mean +/− SEM from n = 6 independent experiments. One sample two-sided t-test. d EVs were isolated from MDA-MB-231-TKS5-GFP-mCh-MT1-MMP cells as described in c, and prepared for electron microscopy as described in the methods. Immunogold labelling using antibodies against mCherry or CD63 show labelling on the surface of the EVs. Graph represents the diameter distribution and the corresponding density of CD63 labelling of in total 239 EVs collected from three independent experiments.
